The role of the yellow stripe-like transporter BdYSL3 in copper homeostasis in Brachypodium

Copper is an essential nutrient for plant growth, development, and reproduction. It plays important roles in photosynthesis, respiration, ethylene perception, and scavenging of reactive oxidative species (Burkhead et al., 2009). Copper deficiency diminishes photosynthesis and pollen fertility which consequently reduces plant growth and seed yield (Huang et al., 2016). Wheat (Triticum aestivum), one of the world’s most important staple foods, is sensitive to copper starvation. Although it has been known for decades that copper deficiency limits wheat crop yields, the underlying mechanisms have remained elusive.

Yellow tripe-ike (YSL) transporters, a subfamily of the oligopeptide (OPT) transporters, have been implicated in the transport of diverse metals, particularly iron and copper. YSL transporters are divided into two classes based on their expression pattern and transport substrates. One class of YSL transporters is unique to and functions in the acquisition of iron (III)–phytosiderophore complexes into the root. The second class is present in both Arabidopsis (Arabidopsis thaliana) and Gramineae and participates in transporting phytosiderophores- or nicotianamine-metal complexes within cells (Curie et al., 2009; Ueno et al., 2009; Chu et al., 2010; Feng et al., 2017). Three of these transporters, AtYSL1, AtYSL3, and OsYSL16, play a role in intracellular copper transport with OsYSL16 and AtYSL3 responsible for copper delivery to pollen, floral organs, and seeds.

In this issue of Plant Physiology, Sheng et al. (2021) investigated the impact of copper deficiency on flower development and fertility by examining the role of transporter YSL3 in wheat and the functional genetics model grass Brachypodium distachyon. They report that B. distachyon YSL3 (BdYSL3) is necessary for proper copper transport through the phloem to reproductive organs. Mutation of BdYSL3 impairs copper delivery to reproductive organs and grains, which delays flowering, alters inflorescence architecture, and reduces floret fertility and grain size. These findings may aid in developing crop plants that exhibit enhanced tolerance to copper deficiency via the manipulation of YSL3 expression or function (Sheng et al., 2021).

The authors first compared growth and fertility of wheat and B. distachyon under copper deficiency. Copper was required for floral and seed development in both species, supporting the use of B. distachyon as a wheat model in copper-deficiency studies. BdYSL3 expression was significantly upregulated by copper starvation in roots, stems, mature leaves, flag leaves, and reproductive organs but not in young leaves where BdYSL3 transcript levels were highest under normal conditions.

Expression of BdYSL3 in Arabidopsis protoplasts or Xenopus oocytes demonstrated plasma membrane localization of BdYSL3, consistent with a role for BdYSL3 in intercellular copper influx/efflux rather than intracellular copper transport. Transport assays with BdYSL3-expressing oocytes demonstrated that BdYSL3 is a bona fide copper transporter that preferentially transports free copper and not copper–nicotianamine complexes, at least in oocytes.

To probe the in vivo role of BdYSL3 in copper transport, the authors generated ysl3 CRISPR/Cas9 mutants in B. distachyon. Mutant plants exhibited a number of abnormal phenotypes, including small shoots, curling leaves, delayed flowering, altered inflorescence, impaired fertility, and smaller grains, demonstrating that BdYSL3 is required for proper growth and flower development. Mutant plants were also more sensitive to copper starvation. Defective inflorescence and fertility of ysl3 mutants were rescued by copper fertilization or complementation with BdYSL3 cDNA, establishing a relation between BdYSL3, copper transport, and reproductive development.

Cell-type-specific expression analysis of BdYSL3 demonstrated the transporter was mainly expressed in the phloem of leaves and nodes, suggesting BdYSL3 functions in internal copper transport rather than acquisition from the soil. Copper quantification experiments showed the ysl3 mutant had lower amounts of copper in the phloem and higher amounts in the xylem compared with wild-type plants, indicating BdYSL3 facilitates copper loading into the phloem. Copper levels in ysl3 plants were elevated in mature leaves and decreased in flag leaves, flowers, and grains compared with wild type. These results further show a role for BdYLS3 in loading copper to phloem and subsequent delivery from mature leaves to sink tissues, including flag leaves, flowers, and grains.

The rice gene OsYSL16 has been previously implicated in the transport of iron as well as copper, so Shen et al. (2021) investigated whether the B. distachyon homolog BdYSL3 plays a role in iron transport. BdYSL3 expression was lower under iron deficiency, and the ysl3 mutant showed no change in iron levels in the flowers or seeds, although higher levels of iron accumulated in roots and flag leaves. In ysl3 mutants, increased iron levels are associated with decreased copper levels. In line with a previous study (Carrió-Seguí et al., 2019), Shen et al. (2021) also showed crosstalk between copper and iron homeostasis. As copper level has a major effect on iron superoxide dismutases (SODs) expression in Arabidopsis and both iron and copper-containing SODs act similarly, the authors suggested increased iron levels may balance the lower copper concentration in ysl3 mutants (Ravet and Pilon, 2013). This evidence suggests BdYLS3 is not involved in iron transport to reproductive organs despite interactions between copper and iron.

This study shows that BdYSL3 encodes a copper transporter that is required for proper copper distribution, similar to its orthologs in rice and Arabidopsis (Chu et al., 2010; Zheng et al., 2012). Specifically, BdYSL3 was required to mobilize copper to flowers and seeds through its function in loading copper to the phloem. In addition, BdYSL3-facilitated copper transport played an important role in flower development and agronomic traits such as grain size and protein accumulation in B. distachyon and wheat. This study also suggests that BdYSL3 primarily transports copper in ionic forms. Since most OPT family members transport metals as a complex with a strong ligand such as nicotianamine or phytosiderophores, future studies should investigate how BdYSL3 functionally cooperates with other copper transporters, including those from and heavy metal transporting P1B-type ATPase families.

Conflict of interest statement. None declared.